This invention refers to cells and organisms for which intermediates of the mevalonate independent isoprenoid metabolism pathway (MEP pathway) are enriched through deletion or inactivation of genes. Furthermore, it refers to processes for producing intermediates and products derived from the MEP pathway from organisms, for which the genes according to the invention have been deleted or inactivated and genetic engineering and convention processes for producing these organisms. It also refers to the application of enzyme inhibitors for enriching MEP pathway intermediates. It also refers to the therapeutic application of cells and organisms for which the genes or enzymes according to the invention have been deleted or inhibited and the production of medication from these cells and organisms.
The biosynthesis of isoprenoids using the classic acetate/mevalonate pathway (Beytia E D, Porter J W, Annu Rev Biochem, 1976; 45: 113-42) and an alternative, mevalonate independent biosynthesis pathway, the 2-methyl-D-erythritol pathway (MEP pathway, synonymous with DOXP pathway) is known (Rohmer M. Nat Prod Rep, 1999 October; 16(5): 565-74). Both pathways lead to isopentenylpyrophosphate (IPP), the common precursor of all higher isoprenoids. While the acetate/mevalonate pathway has been known for some time and is fully understood, at present not all biosynthetic steps in the reaction of the MEP pathway are known.
In the past, various biotechnological processes have been derived, based on the application of knowledge regarding the MEP pathway:
1. Inhibitors of various enzymes through the MEP pathway are suitable as disinfectants and herbicides as the MEP pathway does not occur in humans.
2. Certain intermediates of the MEP pathway lead to a massive stimulation of human gamma/delta T cells. These intermediates are suitable as immunomodular medicines.
3. Through the over-expression of certain genes of the MEP pathway (e.g. DOXP synthase, LytB), the enriching of higher isoprenes can be achieved as subsequent products of the MEP pathway.
It was previously unknown that through the deletion of a gene of the MEP pathway or through inactivation of the corresponding enzyme, an intermediate of the MEP pathway can be achieved.
It is known that human gamma/delta T cells are activated through one or more intermediate of the MEP pathway. This means that with the incubation of peripheral blood lymphocytes with extracts from organisms which have an MEP pathway, there is a selective proliferation and cytokine secretion of the gamma/delta T cell population (Jomaa H, Feurle J, Luhs K, Kunzmann V, Tony H P, Herderich M, Wilhelm m, FEMS Immunol Med. Microbiol. 1999 September; 25(4): 371-8). The exact chemical composition of this activating substance of substances is still unknown. Published data suggest that 3-formyl-1-butylpyrophosphate plays a role as a hypothetical intermediate of the MEP pathway in activating gamma/delta T cells (Belmant C, Espinosa E, Poupot R, Peyrat M A, Guiraud M Poquet Y, Bonneville M Fournte J J, J. Biol. Chem. 1999 Nov. 5; 274(45): 32079-84).
Consequently, it was shown that bacteria, where various genes of the MEP pathway (e.g. DOXP reductoismerase, gepE) had been deleted, were no longer able to activate gamma/delta T cells (Altincicek B, Moll J, Campos N, Foerster G, Beck E, Hoeffler J F, Grosdemange-Billiard C, Rodriguez-Concepcion M, Rohmer M, Boronat A, Eberl M Jomaa H, J. Immunol. 2001 Mar. 15; 166(6):3655-8). In order to produce these deletion mutations it is necessary to introduce genes of the mevalonate pathway using genetic engineering into the bacteria. As a result, the bacteria can then survive in the medium in the presence of mevalonate if the MEP pathway is no longer functional (FIG. 1).